Methods and compositions enhancing survival and functionality of anti-tumor and anti-viral t cells

ABSTRACT

The present invention relates to a method able to enhance survival and functionality of anti-tumor or anti-viral immune cells through overexpression of Akt molecules in the cells. Akt signaling prevented the expression of immune checkpoints and therefore rescued antigen-specific cytotoxic T lymphocytes from exhaustion in immunosuppressive microenvironment. This present invention also demostrated that AKT genes have the potential to be utilized in T-cell engineering of adoptive T-cell therapy for treatment of chronic viral infection and malignancies

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.62/565,820, filed on SEP 29, 2017, the entire content of which is herebyincorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION Technical Field of the Invention

The present invention relates to adoptive cell therapy usingAkt-overexpressing immune cells. More specifically, theAkt-overexpressing immune cells can be utilized for treatment of viralinfection and malignancies in immunosuppressive microenvironment.

Background

Adoptive cell therapy (ACT) utilizing gene engineering to introduceantigen specificity or to enhance effector functions or survival ofimmune cells is feasible and high clinical values for treatment ofchronic infections or malignancies since virus- or tumor-specific immunecell response is usually impaired or missing in patients with most ofthese chronic diseases.

However, during chronic viral infections or malignancies, there areusually monoclonal T cell response detected and most of theantigen-specific T cells undergo exhaustion or apoptosis rapidly afteractivation. It is often observed that the virus or tumor-specificcytotoxicity T lymphocytes (CTLs) undergo T-cell exhaustion due topersistent T-cell receptor (TCR) signaling and lack of suitableco-stimulation. T cell exhaustion features the gradual loss ofproliferative capability and cytokine production, impaired cytotoxicity,surface expression of various immune checkpoints and increase ofapoptotic rate[1, 2].

Immune checkpoints e.g. PD-1 and CTLA-4 are molecules up-regulated on Tcells in response to TCR signaling to modulate the extent of T-cellactivation and are highly expressed on exhausted T cells. It has beenshown in several studies that signaling through immune checkpoints on Tcells can impair metabolic reprogramming during T-cell activation anddifferentiation[3-6].

The molecular pathways by which most of the immune checkpoints signalremain poorly understood except that PP2A and SHP2 activated by PD-1 andCTLA-4 signaling, respectively, can suppress Akt activation of T cellsupon TCR stimulation, being revealed[7].

Akt is shown to have a great influence on T-cell growth, proliferation,and survival and also demonstrated to be a signal integrator for T-celldifferentiation through regulation of Foxo, mTOR and Wnt/β-cateninpathways[8-11]. During chronic LCMV infection, the activation of Akt andmTOR signaling in CTLs is impaired, which results in T-cell exhaustionthrough PD-1 signaling in virus-specific CTLs[12].

Therefore, the present invention demonstrates that reinforcement ofAkt/mTOR pathway in anti-viral or anti-tumor CTLs may rescue them from Tcell exhaustion and has the potential to be further applied onrecombinant TCR technology or chimeric antigen receptor (CAR) technology[13] to enhance the survival and effector functions of engineered Tcells for treatment of patients with malignancy or chronic viralinfection.

SUMMARY OF INVENTION

The present invention provides a method able to enhance survival andfunctionality of anti-tumor or anti-viral T cells through overexpressionof Akt molecules in CTLs. The Akt-overexpressing CTLs are shown to havehigh proliferative capability and superior effector functions duringencounter with the antigen in the liver, which suggests that the Aktmolecules can help the CTLs to overcome T-cell exhaustion in theinhibitory microenvironment. We further show expression of Akt moleculescan facilitate anti-viral and anti-tumor CTL responses e.g.proliferation, cytokine production and cytotoxicity. Moreover, itenables the CTLs resistance to proliferative arrest induced by MDSCs.the expression of constitutively active Akt molecules enable T cells togain the privilege to survive and to kill in the tolerogenic liver ortumor microenvironments. The active Akt molecules only when incombination with TCR signaling can trigger massive proliferativeresponse of CTLs and therefore are safe to be applied to T-cellengineering of CTLs.

In one embodiment, this present invention demonstrates that themyristorylated Akt molecules are able to anchor on cell membrane and canbe phosphorylated. After being adoptive transfer into the recipientmice, Akt1- and Akt2-CTL populations expand vigorously in the liver andthe spleen. It indicates overexpression of Akt is related tointrahepatic survival or secondary expansion of CTLs in response toantigen stimulation.

T cell exhaustion features surface expression of various immunecheckpoints. The immune checkpoint blockade can rescue T cell exhaustionof CTLs and further enhance the anti-tumor responses. In anotherembodiment, this present invention demonstrated that Akt signalingprevents the expression of immune checkpoints, especially LAG-3 andTIGIT on HBV-specific CTLs.

In some embodiments, this present invention demonstrates thatAkt1/2-engineered CTLs clear intrahepatic viral infections efficientlyin two different models and persist and provide protective memoryimmunity in the recovered individuals.

In some embodiments, Akt2-engineered CTLs are able to eradicateestablished liver cancers in an oncogene-induced HCC mouse model. AKT1and AKT2 genes can be utilized in T-cell engineering of adoptive T-celltherapy for treatment of hepatic chronic viral infection andmalignancies since Akt signaling is able to reverse T-cell exhaustion ofCTLs in immunosuppressive microenvironment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1O depict the HBV-specific CTLs undergo T-cell exhaustion afteradoptive transfer into HBV carrier mice. (A) Kinetics of serum HBeAg ofAdHBV-infected mice receiving adoptive transfer of 2×10⁵HBc₉₃₋₁₀₀-specific CTLs. Gating (B) and quantification (C) of CD45.1⁺transferred CTLs in the liver and the spleen of HBV carrier mice atindicated time points post adoptive transfer into AdHBV-infected mice.5×10⁵ in-vitro activated HBc₉₃₋₁₀₀ CD8⁺ T cells are adoptivelytransferred into CD45.2⁺ recipient mice infected with AdHBV. Histogramsshow the expression of PD-1 (D, H, L), TIM-3 (E, I, M) and LAG-3 (F, J,N), on transferred CTLs in the liver and in the spleen of AdHBV-infectedmice from day 3, 7 and 14 post adoptive transfer. The isotype controlstaining is shown in solid gray histogram whereas the specific stainingis shown in open histogram. Mean fluorescence intensity (MFI) of PD-1,TIM-3 and LAG-3 staining on endogenous CD8⁺ T cells and on adoptivelytransferred CD45.1+CD8⁺ T cells (gating as shown in B) in the liver andin the spleen of AdHBV-infected mice from day 3(G), 7 (K) and 14 (O)post adoptive transfer of 5×10⁵ HBc₉₃₋₁₀₀-specific CTLs. **P<0.01 and***P<0.001 (unpaired Student's t-test).

FIGS. 2A-2F depict the regulation of intrahepatic CTL expansion bydifferent Akt isoforms. (A) Schematic representation of MSCV retroviralconstructs used for T-cell engineering contain 5′ and 3′long terminalrepeats (LTR), P2A linker peptide sequence (2A) (SEQ ID NO: 10), CD90.1gene and woodchuck hepatitis virus posttranscriptional regulatoryelement (WPRE). In pMSCV-mAkt1/Akt2/Akt3-2A-CD90.1 plasmid, srcmyristoylation sequence (myr) (SEQ ID NO: 8) and mouse AKT1 (SEQ ID NO:2). AKT2 (SEQ ID NO: 4) or AKT3 (SEQ ID NO: 6) gene are placed upstreamof 2A sequence. (B) Transduction efficiency of in vitro-activatedCD45.1⁺ OT-I cells transduced with retroviruses carrying 2A-CD90.1,mAkt1-2A-CD90.1, mAkt2-2A-CD90.1 and mAkt3-2A-CD90.1, respectively ormock. At day 2 after transduction, the surface expression of CD90.1 as amarker for successful transduction is detected by flow cytometricanalysis. (C) Western blot for detection of phospho-Akt, total Akt,β-actin and phospho-S6 proteins in the cell lysate of ctrl, Akt1, Akt2and Akt3-transduced CD8⁺ T cells. (D) Quantification of transferred CTLsin the liver and spleen of the mice with intrahepatic expression of thecognate antigen. 1×10⁵ transduced OT-I CTLs are adoptively transferredinto recipient mice receiving hydrodynamic injection (HDI) of a plasmidencoding ovalbumin and luciferase under the control of albumin promoterone day before adoptive transfer. The liver-associated lymphocytes andsplenocytes are isolated at day 7 after adoptive transfer and subjectedto flow cytometric analysis of the percentage and the number of thetransferred CTLs. Kinetics of accumulation of transferred CTLs in theliver (E) and spleen (F) of the mice with intrahepatic expression of thecognate antigen (ovalbumin). 1×10⁵ transduced OT-I CTLs are adoptivelytransferred into recipient mice receiving HDI of a plasmid encodingovalbumin and luciferase under the control of albumin promoter one daybefore adoptive transfer. The liver-associated lymphocytes andsplenocytes are isolated at day 3, 7 and 15 and subjected to flowcytometric analysis of the percentage and the number of the transferredCTLs. *P<0.05, **P<0.01 and ***P<0.001 (unpaired Student's t-test)

FIGS. 3A-3B depict the local expansion of Akt2-engrafted OT-I CTLs. (A)Kinetics of hepatic in vivo bioluminescence in mice receiving HDI of aplasmid encoding OVA under the control of albumin promoter or a ctrlvector (ctrl) one day before adoptive transfer of 2A-luc-engineered(ctrl) OT-I or mAkt2-2A-luc-engineered (Akt2) OT-I cells. Thebioluminescence of individual mouse is monitored at day 1, 4, 8, 10, 12,15, 18 and 25 after adoptive transfer and plotted in (B).

FIGS. 4A-4T depict the Akt-engineered HBc₉₃₋₁₀₀-specific CTLs overcameT-cell exhaustion in the liver. Histograms of expression of PD-1 (A),TIGIT (B) and LAG-3 (C) on Akt1-CD90.1- or CD90.1-engineered (ctrl) CTLsbefore adoptive transfer. The isotype control staining is shown in solidgray histogram whereas the specific staining is shown in open histogram.(D) Mean fluorescence intensity (MFI) of the staining results from A-Cis shown in bar graph. (E) PD-1, (F) TIGIT and (G) LAG-3 onCD90.1-engineered (ctrl) CTLs and Akt1-CD90.1-engineered CTLs after24-hours re-stimulation with anti-CD3/CD28 beads. The isotype controlstaining is shown in solid gray histogram whereas the specific stainingis shown in open histogram. (H) MFI of the staining results from E-G isshown in bar graph. 5×10⁵ Akt1-CD90.1- or CD90.1-engineered (ctrl) areadoptively transferred into CD45.2⁺ recipient mice being infected withAdHBV. The liver-associated lymphocytes and splenocytes are isolated atday 6 or day 19 post adoptive transfer and subjected to flow cytometricanalysis of the expression of immune checkpoints by the transferredCTLs. CD8⁺ CD45.1⁺ cells are gated and defined as transferred CTLs.Expression of immune checkpoints, PD-1 (I, J), TIM-3 (M, N) and LAG3 (Q,R), on transferred CTLs from day 6 post adoptive transfer. Expression ofimmune checkpoints, PD-1 (K, L), TIM-3 (O, P) and LAG3 (S, T), ontransferred CTLs from day 19 post adoptive transfer. The isotype controlstaining is shown in solid gray histogram whereas the specific stainingis shown in open histogram. MFI of the staining results are shown in J,L, N, P, R and T. (n=3 per group). *P<0.05, **P<0.01 and ***P<0.001(unpaired Student's t-test)

FIGS. 5A-5H depict the influence of Akt signaling in the expression ofimmune checkpoints in vitro. Histograms of expression of (A) PD-1, (B)TIGIT and (C) LAG-3 on CD90.1-engineered (ctrl) CTLs, Akt1-CD90.1- andAkt2-CD90.1 CTLs after 3-days stimulation with anti-CD3/CD28 beads. Theisotype control staining is shown in solid gray histogram whereas thespecific staining is shown in open histogram. (D) MFI of the stainingresults from A-C is shown in bar graph. Histograms of expression of (E)PD-1, (F) TIGIT and (G) LAG-3 on CD90.1-engineered (ctrl) CTLs andAkt2-CD90.1-engineered CTLs after 24-hours re-stimulation withanti-CD3/CD28 beads. The isotype control staining is shown in solid grayhistogram whereas the specific staining is shown in open histogram. (H)MFI of the staining results from E-G is shown in bar graph. *P<0.05,**P<0.01 and ***P<0.001 (unpaired Student's t-test)

FIGS. 6A-6F depict the Akt2-engineered HBc₉₃₋₁₀₀-specific CTLs preventT-cell exhaustion in a persistent HBV mouse model. 2×10⁶ Akt2-CD90.1- orCD90.1-engineered (ctrl) is adoptively transferred into CD45.2+recipient mice infected with AdHBV. The liver-associated lymphocytes andsplenocytes are isolated at day 19 post adoptive transfer and subjectedto flow cytometric analysis of the expression levels of immunecheckpoints. Histograms of expression of PD-1 (A), TIM-3 (C) and TIGIT(E) on transferred CTLs in the spleen or liver of recipient mice at day19 post adoptive transfer. The isotype control staining is shown insolid gray histogram whereas the specific staining is shown in openhistogram. MFI of the staining results is shown in B, D and F. (n=3 pergroup). *P<0.05, **P<0.01 and ***P<0.001 (unpaired Student's t-test)

FIGS. 7A-7O depict the Akt-engineered HBc₉₃₋₁₀₀-specific CTLs developedprotective immunity against HBV in a persistent HBV mouse model. Gating(A) and quantification (B, C) of CD45.1⁺ transferred CTLs in the liverand the spleen of HBV carrier mice at day 6 (B) or day 19 (C) postadoptive transfer into AdHBV-infected mice. 5×10⁵ Akt1-CD90.1- orCD90.1-engineered (ctrl) are adoptively transferred into CD45.2⁺recipient mice being infected with AdHBV 2.5 months ago. Theliver-associated lymphocytes and splenocytes are isolated at day 6 orday 19 post adoptive transfer and subjected to flow cytometric analysisof the percentage and the number of the transferred CTLs. CD8⁺ CD45.1⁺cells are gated and defined as transferred CTLs. (D) Kinetics of serumHBeAg of recipient mice as in C. (E) Kinetics of serum ALT of recipientmice as in C. (F) Hematoxylin-and-eosin staining of the liver tissuesfrom B. Immunohistochemical analysis of HBcAg (G), cleaved caspase 3(H), Gr-1 (I) and CD45.1 (J) in the liver from B. (K)Hematoxylin-and-eosin staining of the liver tissues from C.Immunohistochemical analysis of HBcAg (L), cleaved caspase 3 (M), Gr-1(N) and CD45.1 (O) in the liver from C. (n=3-4 per group). *P<0.05,**P<0.01 and ***P<0.001 (unpaired Student's t-test). Scale bars, 100 or40 μm.

FIGS. 8A-8D depict the Akt2-engineered HBc₉₃₋₁₀₀-specific CTLs developeprotective immunity against HBV in a persistent HBV mouse model. Gating(A) and quantification (B) of CD45.1⁺ transferred CTLs in the liver andthe spleen of HBV carrier mice at day 19 post adoptive transfer intoAdHBV-infected mice. 2×10⁶ Akt2-CD90.1- or CD90.1-engineered (ctrl)HBc₉₃₋₁₀₀-specific CTLs are adoptively transferred into CD45.2⁺recipient mice infected with AdHBV. The liver-associated lymphocytes andsplenocytes are isolated day 19 post adoptive transfer and subjected toflow cytometric analysis of the percentage and the number of thetransferred CTLs. CD8⁺ CD45.1⁺ cells are gated and defined astransferred CTLs. (C) Kinetics of serum ALT of recipient mice as in B.(D) Kinetics of serum HBeAg of recipient mice as in B. *P<0.05, **P<0.01and ***P<0.001 (unpaired Student's t-test)

FIGS. 9A-9D depict the cytokine production in HBV-specific CTLs afteradoptive transfer into HBV carrier mice. (A) Zebra plots ofintracellular expression of IFN-γ and TNF-α in adoptively transferredHBV-specific CTLs. 5×10⁵ Akt1-CD90.1- or CD90.1-engineered (ctrl)HBc₉₃₋₁₀₀-specific CTLs are adoptively transferred into CD45.2⁺recipient mice infected with AdHBV. The liver-associated lymphocytes andsplenocytes are isolated at day 19 post adoptive transfer and subjectedto re-stimulation with HBc₉₃₋₁₀₀ peptides for 6 hours, which is followedby staining of surface markers and intracellular cytokines and flowcytometric analysis of the percentage of the cytokine-secreting CTLs.CD8⁺ CD45.1⁺ cells are gated and defined as transferred CTLs. (B) Bargraph of the percentage of IFN-γ-secreting CTLs (SP) and the percentageof CTLs secreting both IFN-γ and TNF-α (DP). (C) Zebra plots ofintracellular expression of IFN-γ and TNF-α in adoptively transferredHBV-specific CTLs. 5×10⁵ Akt2-CD90.1- or CD90.1-engineered (ctrl)HBc₉₃₋₁₀₀-specific CTLs are adoptively transferred into CD45.2⁺recipient mice infected with AdHBV. The liver-associated lymphocytes andsplenocytes are isolated at day 19 post adoptive transfer and subjectedto re-stimulation with HBc₉₃₋₁₀₀ peptides for 6 hours, which is followedby staining of surface markers and intracellular cytokines and flowcytometric analysis of the percentage of the cytokine-secreting CTLs.CD8⁺ CD45.1⁺ cells are gated and defined as transferred CTLs. (D) Bargraph of the percentage of IFN-γ-secreting CTLs (SP) and the percentageof CTLs secreting both IFN-γ and TNF-α (DP). *P<0.05, **P<0.01 and***P<0.001 (unpaired Student's t-test)

FIGS. 10A-10J depict the Akt signaling facilitates antigen-dependentexpansion of CTLs and the antigen clearance in the liver. (A) Thepercentage of bioluminescence-positive mice equivalent to OVA-positivemice at indicated time points. Kinetics of accumulation of transferredCTLs in the liver (B) and spleen (C) of the mice with intrahepaticexpression of the cognate antigen (ovalbumin). 1×10⁵ transduced OT-ICTLs were adoptively transferred into recipient mice receivinghydrodynamic injection (HDI) of a plasmid encoding ovalbumin andluciferase under the control of albumin promoter one day before adoptivetransfer. The liver-associated lymphocytes and splenocytes were isolatedat day 3, 7 and 14 and subjected to flow cytometric analysis of thepercentage and the number of the transferred CTLs. (D) Kinetics of serumALT in OVA-Luc-positive mice receiving adoptive transfer of 1×10⁵2A-CD90.1-engrafted (ctrl) or mAkt1-2A-CD90.1-engrafted (Akt1) OT-Icells. (E, F) Kinetics of accumulation of transferred CTLs in the liver(E) and spleen (F) of the mice as in A. The liver-associated lymphocytesand splenocytes were isolated at day 7, 30 and 63 and subjected to flowcytometric analysis of the percentage and the number of the transferredCTLs. (G) Hematoxylin-and-eosin staining of the liver tissues from E.(H) A representative histogram of BrdU-staining of Akt1-engrafted OT-ICTLs at day 7 and day 63 after adoptive transfer into OVA-Luc-positiverecipient mice. (I) Frequency of BrdU⁺ transferred Akt1-engrafted OT-ICTLs at day 7 and day 63 after adoptive transfer into OVA-Luc-positiverecipient mice. The recipient mice were given 1 mg BrdU viaintraperitoneal injection at day 6 or day 62 after adoptive transfer.The liver-associated lymphocytes and splenocytes were isolated at day 7and 63 and subjected to flow cytometric analysis of the percentage theBrdU⁺ transferred CTLs. (J) Immunohistochemical analysis of Ki-67 in theliver of OVA-Luc-positive mice receiving adoptive transfer of2A-CD90.1-engrafted (ctrl) or mAkt1-2A-CD90.1-engrafted (Akt1) OT-Icells. The liver was collected at day 7, day 32 and day 63 afteradoptive transfer of 1×10⁵ OT-I CTLs. Scale bars, 40 μm. *P<0.05,**P<0.01 and ***P<0.001 (unpaired Student's t-test), Scale bars, 100 or40 μm.

FIGS. 11A-11B depict in vivo bioluminescence of mice infected withAd-Albp-OL. C57BL/6 mice are infected with a recombinant adenoviruscarrying genes expressing ovalbumin and luciferase under the control ofalbumin promoter at different viral doses. The infected mice aremonitored for the luciferase expression in the liver by IVIS atindicated time points after infection.

FIGS. 12A-G depict the memory responses of Akt-engineered CD8⁺ T cells.(A) Experimental scheme of re-call response of Akt-engrafted CTLs. (B)The level of serum ALT in the mice receiving adenovirus carrying OVA andluciferase ORFs under the control of albumin promoter (Ad-Albp-OL) andcontrol (ctrl) or Akt1-engrafted OT-I T cells (1×10⁵) at indicated timepoints post adoptive T cell transfer. (C) The in vivo bioluminescence inmice receiving Ad-Albp-OL, adoptive T cell transfer and hydrodynamicinjection (HDI) of a plasmid encoding OVA and luciferase under thecontrol of albumin promoter (pENTRY-Albp-OL) at day 60 after adoptivetransfer. (D) Quantification of transferred CTLs in the liver and spleenof the mice receiving Ad-Albp-OL infection and adoptive transfer of ctrl2A-CD90.1 engrafted OT-I or Akt1-engrafted OT-I followed by HDI ofpENTRY-Albp-OL at day 60 after adoptive transfer. The liver-associatedlymphocytes and splenocytes were isolated at day 7 after HDI andsubjected to flow cytometric analysis of the number of the transferredCTLs. (E) Hematoxylin-and-eosin staining of the liver tissues from D.(F) Immunohistochemical analysis of CD8 in the liver from D. (G)Immunohistochemical analysis of Gr-1 in the liver from D. *P<0.05,**P<0.01 and ***P<0.001 (unpaired Student's t-test), Scale bars, 100 or40 μm.

FIGS. 13A-13F depict the memory responses of Akt-engineered CD8⁺ Tcells. Mice are infected with Ad-Albp-OL, and receive adoptive T celltransfer and HDI of a plasmid encoding OVA and luciferase under thecontrol of albumin promoter (pENTRY-Albp-OL) at day 64 after adoptivetransfer. Quantification of (A) transferred CTLs, (B) CD11b⁺NK1.1⁻myeloid cells, (C) NK1.1⁺ CD3⁻ NK cells and (D) NK1.1⁺ CD3⁺ NKT cells inthe liver and spleen of the mice receiving Ad-Albp-OL infection andadoptive transfer of ctrl 2A-CD90.1, Akt1 or Akt2 engrafted OT-I CTLs.The liver-associated leukocytes and splenocytes are isolated at day 7after adoptive transfer and subjected to flow cytometric analysis of thenumber of cells. (E) The level of serum ALT in the mice receivingAd-Albp-OL and ctrl or Akt2-engrafted OT-I T cells (1×10⁵) at indicatedtime points post adoptive T cell transfer. (F) The in vivobioluminescence in mice receiving Ad-Albp-OL, adoptive T cell transferand HDI of a plasmid encoding OVA and luciferase under the control ofalbumin promoter (pENTRY-Albp-OL) at day 64 after adoptive transfer.*P<0.05, **P<0.01 and ***P<0.001 (unpaired Student's t-test)

FIGS. 14A-14C depict the influence of Akt-engineered CTLs in HCC tumormicroenvironment. Immunohistochemical analysis of CD8 (A), F4/80 (B),and cleaved caspase 3 (C) in the liver/tumor of HCC-bearing mice. TheHCC development is induced by oncogenes, Akt and N-RasV12 delivered byHDI. At day 31 after HCC induction, the mice are injected with 2×10⁶Akt2-engrafted OT-I TCR tg CTLs which could recognize an introducedtumor antigen on tumor cells or not (ctrl). The liver/tumor tissues arecollected at day 10 after adoptive transfer.

FIGS. 15A-15D depict the anti-tumor capability of Akt-engineered CTLs.The HCC development is induced by oncogenes, Akt and N-RasV12 deliveredby HDI. The growth of HCC in mice is monitored by IVIS and the mice withthe total flux greater than 10⁹ photons/sec are used as recipientsreceiving adoptive T cell therapy. The mice are injected with 2×10⁵ctrl-, Akt1- and Akt2-engrafted HBc₉₃₋₁₀₀-specific CTLs, respectively,which can recognize a surrogate tumor antigen on tumor cells. (A) The invivo bioluminescence of the mice before and after receiving adoptive Tcell transfer. The liver/tumor tissues are collected from mice receiving(B) ctrl-engineered CTLs, (C) Akt1-engineered CTLs or (D)Akt2-engineered CTLs at day 19 after adoptive transfer. *P<0.05,**P<0.01 and ***P<0.001 (unpaired Student's t-test)

FIGS. 16A-16L depict the improved tumor-specific proliferation, cytokineproduction and cytotoxicity of CAR T cells through overexpression of Aktmolecules. (A) Schematic representation of MSCV retroviral constructsused for T-cell engineering contain 5′ and 3′long terminal repeats(LTR), P2A linker peptide sequence (2A) and woodchuck hepatitis virusposttranscriptional regulatory element (WPRE). InpMSCV-mAkt1/Akt2-2A-CAR plasmid, src myristoylation sequence (myr) andmouse AKT1 or AKT2 gene are placed upstream of 2A sequence, followed bychimeric antigen receptor (CAR) ORF e.g. anti-HBs CAR (S-CAR) andanti-CEA CAR In pMSCV-hAkt1/hAkt2-2A-CAR plasmids, the mouse AKT1 orAKT2 gene is replaced by human AKT1 or AKT2 gene. (B) Proliferation ofAkt1-engrafted (mAkt1), anti-CEA CAR-engrafted (antiCEA) andAkt1-2A-anti-CEA CAR (mAkt1-antiCEA) CD4⁺ or CD8⁺ T cells. Invitro-activated mouse CD3⁺ T cells transduce with retroviruses carryingmAkt1/mAkt2-2A-CD90.1, anti-CEA CAR or mAkt1/mAkt2-2A-anti-CEA CAR ORF,respectively are co-cultured with LS174T cells. EdU incorporation anddetection are applied to monitor the DNA synthesis of the T cells during22 hours to 28 hours after co-culture. (C, E) IFNγ and (D, F) IL-2 inthe supernatant of the co-culture are detected by ELISA. (G, I)Intracellular IFNγ and (H, J) granzyme B staining of the CTLs from theco-culture with LS174T cells for 1 day. (K) Proliferation capability ofCTLs in the presence of MDSCs. 2A-CD90.1-engrafted (ctrl) ormAkt1-2A-CD90.1-engrafted OT-I CTLs are re-stimulated withanti-CD3+anti-CD28 beads in the presence of different numbers of MDSCsderived from EL4-tumor-bearing mice. (L) Proliferation capability ofCTLs in the presence of MDSCs. 2A-CD90.1-engrafted (ctrl) ormAkt2-2A-CD90.1-engrafted HBc₉₃₋₁₀₀ specific CTLs are re-stimulated withanti-CD3+anti-CD28 beads in the presence of different numbers of MDSCsderived from mouse HCC tumor mass. EdU incorporation and detection areperformed to monitor the DNA synthesis of the T cells during 22 hours to28 hours after co-culture. *P<0.05, **P<0.01 and ***P<0.001 (unpairedStudent's t-test)

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by a person skilled in theart to which this invention belongs.

As used herein, the term “OT-I cell” refers to a transgenic line ofovalbumin-specific, CD8⁺ T cell. The transgenic T cell receptor wasdesigned to recognize ovalbumin residues 257-264 in the context ofH-2K^(b) and used to study the role of peptides in positive selectionand the response of CD8⁺ T cells to antigen.

As used herein, the term “AdHBV” refers to the adenovirus carrying HBVgenome. HBV-infected mouse model can be established by hydrodynamicinjection (HDI) of the HBV genome into the tail vein.

As used herein, the term “HBcAg” refers to a hepatitis B viral protein,which is an antigen that can be found on the surface of the nucleocapsidcore of the hepatitis B virus.

As used herein, the term “HBeAg” refers to a hepatitis B viral protein,which is an antigen that can be detected in the serum of mice with HBVinfection established by AdHBV infection or HDI of a plasmid harboringthe HBV genome.

The DNA or RNA molecules in this present invention can be amplifiedthrough plasmid amplification, in vitro transcription or in vitrosynthesis and transfected into target cells through electroporation,liposome or other chemical vehicles.

The aforementioned target cells for genetic modification can be T cells,nature killer cells, hematopoietic stem cells, embryonic stem cells andpluripotent stem cells from various species. These cells can be modifiedby viral transduction or DNA (or RNA) transfection.

The recombinant viral or transposon vectors can be retroviruses,lentiviruses, adenoviruses, adeno-associated viruses, other relatedviruses and various transposon systems can be used in transduction orintegration of transgenes.

To investigate the mechanism of how liver microenvironment can influencesecondary expansion of virus-specific CTL population in the liver, invitro-activated HBV specific CD8⁺ T cells are adoptively transferredinto HBV carrier mice and the change of the serum level of HBV antigenin these mice is detected. It is found that most of the mice failed toeliminate persistent HBV infection within 42 days. The cell number andexpression level of exhaustion markers including PD-1, TIM-3, and LAG-3on the adoptively transferred CTLs in the liver and in the spleen of theHBV carrier mice are further detected. The cell number of adoptivelytransferred HBV-specific CTLs increases in the liver but not in thespleen. The HBV-specific CTLs in both the liver and the spleen expresshigher levels of PD-1 and LAG-3 than endogenous CD8⁺ T cells; however,the splenic HBV-specific CTLs express lower levels of PD-1. TIM-3 andLAG-3 than intrahepatic compartments. Those results demonstrate that theexposure to HBV antigens expressed in the liver microenvironment inducesT-cell exhaustion of HBV-specific CTLs.

The immune checkpoints PD-1 and CTLA-4 are shown to prevent Aktphosphorylation % activation during TCR triggering through recruitmentof SHP-1/2 and activation of PP2A, respectively. We therefore examinewhether Akt signaling is critical to intrahepatic expansion anddifferentiation of CD8⁺ T cells. Mouse AKT1, AKT2 and AKT3 genes arecloned, respectively, with addition of src myristoylation sequence inthe upstream of AKT genes to ensure the membrane targeting and beingconstitutively active of the Akt molecules. The expression of exogenousmyristoylated Akt isoforms are detected by Western blot inAkt-engineered CTLs but not in the control T cells. CTLs are engraftedwith three different kinds of Akt, respectively, all show Aktphosphorylation at Ser473 and only those are engrafted with Akt1 or Akt2show Akt phosphorylation at Thr308.

To examine whether overexpression of Akt is related to intrahepaticsurvival or secondary expansion of CTLs in response to antigenstimulation, the ovalbumin (OVA) and luciferase expression are inducedin the liver of recipient mice by hydrodynamic injection (HDI) of aplasmid encoding OVA and luciferase. After being adoptive transfer intothe recipient mice, Akt1- and Akt2-engineered CTL populations expandvigorously in the liver and the spleen. There is more than 250,000-foldfor Akt1 CTLs and 950,000-fold for Akt2-CTLs cell numbers found in theliver in comparison with that of ctrl-CTLs at day 7 after adoptivetransfer.

Owing to the huge contribution of immune checkpoints on T-cellexhaustion in the liver during chronic viral infection, the inventorstherefore examine whether Akt signaling have an influence the expressionof immune checkpoint molecules on HBV-specific CTLs per se. Afterin-vitro activation and transduction, the Akt- or ctrl-engineeredHBc₉₃₋₁₀₀-specific CTLs are adoptively transferred into AdHBV-infectedmice and analyzed the surface expression of immune checkpoint moleculeson the CTLs at day 6 and day 19 after adoptive transfer. Hepaticctrl-CTLs expressed high level of PD-1, TIM-3 and LAG-3 at day 19 afteradoptive transfer, whereas Akt1-CTLs and Akt2-CTLs expressedsignificantly less PD-1, TIM-3 and LAG-3 at day 19 post adoptivetransfer.

To further investigate whether these Akt-CTLs can overcome thesuppressive mechanisms in the liver and mediate clearance of persistentHBV infection, the ctrl- or Akt1-engineered HBc₉₃₋₁₀₀-specific CTLs areadoptively transferred into HBV carrier mice. Akt1-CTLs but notctrl-CTLs eliminate persistent HBV infection within 14 days after beingadoptive transferred into HBV carrier mice. The Akt1-CTLs are mainly inthe liver rather than in the spleen and disperse to the spleen afterantigen clearance. There are less HBcAg-positive hepatocytes but morecleaved caspase 3-positive apoptotic hepatocytes detected in the liverof mice receiving Akt1-CTLs than in the liver of mice receivingctrl-CTLs. After clearance of antigen, the mononuclear cells reduce andHBcAg-positive hepatocytes as well as cleaved caspase 3-positivehepatocytes are no longer detected in the liver of mice receivingAkt1-CTLs. The ctrl-CTLs fail to clear HBV and do not induce significantinflammation after being adoptively transferred into HBV carrier mice.Akt2-CTLs expand vigorously when encountering the cognate antigen invivo, and prevent T-cell from exhaustion. Also, Akt2-CTLs exhibit strongcytotoxic function and are more efficient to clear HBV infection thanctrl CTLs.

The capability of Akt-engineered CTLs in killing of hepatocellularcarcinoma (HCC) is further examined. The tumor antigen-specificAkt2-engrafied CD8⁺ CTLs can accumulate in the tumor sites as well as inthe liver at day 10 after adoptive transfer into HCC-bearing mice. TheseAkt2-CTLs change the tumor microenvironment and to attract or activatethe surrounding F4/80⁺ macrophages in tumor sites. Furthermore, a lot ofcleaved caspase 3-positive tumor cells are detected in the micereceiving Akt2-CTLs but not in ctrl mice. Elevated serum ALT in the micewith Akt2-CTLs is also observed but not in ctrl mice (118.1 U/L vs. 22.8U/L). It can be concluded that Akt2 activation enables CTLs to havestrong effector functions and be able to kill tumor cells in the liver.This is probably through CTLs' own cytotoxic capability or throughrelease of cytokines to activate the anti-tumor functions oftumor-associated macrophages.

To further explore the potential application of Akt molecules on cancerimmunotherapy, the plasmids carrying human or mouse Akt1 or Akt2 genesand anti-CEA (Carcinoembryonic antigen) chimeric antigen receptor (CAR)are constructed. CEA are glycosyl phosphatidyl inositol (GPI)cell-surface-anchored glycoproteins and are critical to thedissemination of colon carcinoma cells. The modified CTLs areco-cultured with a colorectal adenocarcinoma cell line, LS174T. BothCD4⁺ and CD8⁺ T cells with the engraftment of anti-CEA CAR can respondto stimulation of LS174T and proliferate. Additional active Akt1expression in anti-CEA CAR engrafted T cells can promote theproliferation capability of both CD4⁺ and CD8⁺ T cells. More IL-2 andIFNγ are detected in the culture medium of co-culture of LS174T cellline with T cells expressing anti-CEA CAR and Akt1 or Akt2 moleculescompared to that of LS174T and T cells expressing solely anti-CEA CAR.Intracellular staining of IFNγ and granzyme B of the CD8⁺ T cellsco-culture with LS174T cells also proves that Akt1 or Akt2overexpression can enhance the cytokine production and cytotoxicity inCTLs. Strikingly, Akt1- and Akt2-overexpressing CTLs, respectively areshown to have the capability to overcome the proliferative arrestinduced by myeloid-derived suppressor cells (MDSCs), which stronglysuggests that the potential application of Akt molecules on T-cellengineering technology e.g. CAR T cells for immunotherapy.

The following examples are offered by way of illustration and not by wayof limitation. The mAkt isoforms are utilized in the mouse model as ademonstration in this present invention, but is not intended to limitthe scope of the invention.

Example 1: Cytotoxic T Lymphocytes Undergo Exhaustion in the Liver

In vitro-activated CD45.1 HBc₉₃₋₁₀₀ specific CD8⁺ T cells are adoptivelytransferred into congenic C57BL/6 mice infected with the adenoviruscarrying HBV genome (AdHBV), and the change of the serum level of HBeAgin these mice is detected. It is found that most of the mice failed toeliminate persistent HBV infection within 42 days (FIG. 1A).

The cell number and expression level of exhaustion markers are furtherdetected, which including PD-1, TIM-3, and LAG-3 on the adoptivelytransferred CTLs in the liver and in the spleen of the HBV carrier miceat day 3, day 7 and day 14 post adoptive transfer. The cell number ofadoptively transferred HBV-specific CTLs increases from day 3 to day 14in the liver but not in the spleen (FIGS. 1B and 1C).

Endogenous CD8⁺ T cells are used as a reference population forevaluation of the expression level of these exhaustion markers onHBV-specific CTLs. The HBV-specific CTLs in both the liver and thespleen express higher levels of PD-1 and LAG-3 than endogenous CD8⁺ Tcells but no or little TIM-3 at day 3 and day 7 post adoptive transfer(FIGS. 1D-1K).

The splenic HBV-specific CTLs express lower levels of PD-1 and LAG-3than intrahepatic compartments at all time points (FIGS. 1D-1O). TheHBV-specific CTLs gradually express TIM-3 after adoptive transfer andreach to a higher level of expression than endogenous CD8⁺ T cells atday 14 in the liver but not the spleen (FIGS. 1E, 1G, 1I, 1K, 1M and1I).

Example 2: Expression of Constitutively Active Akt Isoforms in CTLs

Murine stem cell retroviral (MSCV) system is chosen for delivery ofgenes into T lymphocytes due to its high efficiency to transducehematopoietic cell lineages. A pMSCV-CD90.1 plasmid is generated from areplacement of hygromycin resistance gene by p2A peptide sequence andmouse CD90.1 open reading frame (ORF) with the woodchuck hepatitis virusposttranscriptional regulatory element (WPRE) in the 3′ untranslatedregion of CD90.1 gene to enhance the expression of the transgenes. TheCD90.1 gene and WPRE sequence are amplified from pLKO_TRC024 plasmid(RNAi core lab, Taipei, Taiwan). Mouse AKT1 (SEQ ID NO: 1). AKT2 (SEQ IDNO: 3) and AKT3 (SEQ ID NO: 5) genes are cloned, respectively, throughPCR using cDNA from mouse 4T1 breast cancer cells with addition of srcmyristoylation sequence by PCR primer in the upstream of AKT genes toensure the membrane targeting and being constitutively active of the Aktmolecules. The myristoylation sequence and AKT genes are linked,respectively, to mouse CD90.1 gene by p2A peptide sequence inpMSCV-CD90.1 to result in pMSCV-mAkt1-CD90.1, pMSCV-mAkt2-CD90.1 andpMSCV-mAkt3-CD90.1. The expression cassette is flanked by 5′ and 3′ MSCVlong terminal repeats (LTRs). The 4 plasmids are used to producerecombinant retroviruses carrying mouse AKT1. AKT2, AKT3 or controlCD90.1 gene, respectively (FIG. 2A).

Splenic ovalbumin-specific TCR tg OT-I CD8⁺ T cells are activated byanti-CD3+anti-CD28 beads, subsequently transduced by recombinantretroviruses and are subjected to surface marker staining using antibodyrecognizing CD90.1 as a tag for transgene expression followed by flowcytometric analysis. Around 75% to 95% of the effector CD8⁺ T cells aretransduced with retroviruses carrying CD90.1, AKT1-CD90.1 or AKT2-CD90.1gene, positive for CD90.1, whereas only 23% of the cells are transducedwith retroviruses carrying AKT3-CD90.1 gene expressed low level ofCD90.1 (FIG. 2B).

It has been shown that the expression patterns of the three Akt isoformsare different. Akt1 (SEQ ID NO: 1) and Akt2 (SEQ ID NO: 3) areubiquitously expressed in nearly all tissues whereas Akt3 (SEQ ID NO: 5)are mainly expressed in brain and testes. The tissue specific expressionmanner of Akt isoforms may explain the low expression of Akt3 by theCD8⁺ T cells. The expression of exogenous myristoylated Akt isoforms isdetected by Western blot in Akt-engineered CTLs but not in the control Tcells. CTLs engrafted with three different kinds of Akt, respectively,all show Akt phosphorylation at Ser473 and only those which areengrafted with Akt1 or Akt2 show Akt phosphorylation at Thr308 (FIG.2C).

Example 3: Akt Signaling Facilitates Antigen-Dependent Expansion of CTLsin the Liver

Ovalbumin (OVA) and luciferase expression are induced in the liver ofrecipient mice by hydrodynamic injection (HDI) of a plasmid encoding OVAand luciferase under the control of albumin promoter (pENTRY-Albp-OL).After being adoptive transfer into the recipient mice, Akt1- and Akt2-but not Akt3-engineered CTL or CD90.1-engineered (ctrl) populationsexpanded vigorously in the liver and the spleen.

These Akt1- or Akt2-CTLs underwent vigorous proliferation and yielded 23million (Akt1) and 113 million (Akt2) splenic and intrahepatic CTLs intotal, respectively, after antigen stimulation in the liver (FIG. 2D)despite that there only 0.1 million activated CD8⁺ T cells areoriginally injected into the recipient mice. Most of the ctrl CTLsdisappear after adoptive transfer probably due to the lack ofco-stimulation, growth signals or the suppressive livermicroenvironment.

The massive expansion of Akt1- or Akt-2-OT-I CTLs is further confirmedin a time kinetic experiment (FIGS. 2E and 2F). Akt2-CTLs are found tobe more potent in expansion in the liver and in the spleen than ctrl- orAkt1-CTLs (FIGS. 2D-F). Moreover, Akt1-CTLs preferentially locate in theliver rather than the spleen (FIGS. 2D-F).

Therefore, Akt constructs with co-expression of luciferase instead ofCD90.1 are designed for monitoring the distribution and expansion ofAkt-engineered CTLs. Control (ctrl) Luc-CTLs and Akt2-Luc-CTLs aredelivered respectively, to mice with or without OVA expression in theirlivers and only observed TCR signaling-dependent Akt2-Luc-CTLaccumulation in the liver but not in other organs or in mice withoutantigen expression in the liver (FIGS. 3A-3B), which suggests thatsignaling through constitutively active Akt can assist massive CTLexpansion only in combination with TCR triggering and these Akt-CTLsundergo T-cell contraction after the clearance of antigen. Again, thectrl CTLs fail to expand in respond to antigen stimulation in the liver(FIGS. 3A-3B).

Example 4: Akt Signaling Suppresses the Expression of Immune CheckpointMolecule on CTLs

After in-vitro activation and transduction, HBc₉₃₋₁₀₀-specific CD8⁺ Tcells at day 3 after activation are analyzed for their surfaceexpression of various immune checkpoints. The overexpression ofconstitutively active Akt12 does not change the surface expression ofPD-1 and TIGIT (FIGS. 4A, 4B and 4D, FIGS. SA, SB and SD): however, itsignificantly reduces the expression of LAG-3 on the surface of Akt1-and Akt2-CTLs (FIGS. 4C and 4D, FIGS. 5C and 5D).

These CTLs at day 3 after anti-CD3/anti-CD28 bead activation may havereturned to resting status with low or no expression of immunecheckpoints e.g. PD-1 and TIGIT except LAG-3. Therefore, the expressionlevel of these immune checkpoints on CTLs after re-stimulation ismeasured. Expression of PD-1 is rapidly detected on ctrl-, Akt1- andAkt2-CTLs (FIGS. 4E and 4H, FIGS. SE and 5H) and slightly higher onAkt1-CTLs than ctrl-CTLs (FIGS. 4E and 4H). However, the expression ofPD-1 on Akt2-CTLs is lower than ctrl-CTLs (FIGS. SE and 5H). Notably,the Akt1- or Akt2-CTLs maintain relatively lower expression of LAG-3 andTIGIT than ctrl-CTLs after re-stimulation with anti-CD3/CD28 beads for24 hours (FIGS. 4F-H, FIGS. 5F-H).

To further investigate whether the regulation of immune checkpoints onCTLs by Akt signaling also happens in liver microenvironment, the Akt1-or ctrl-engineered HBc₉₃₋₁₀₀-specific CTLs are adoptively transferredinto AdHBV-infected mice and analyzed the surface expression of immunecheckpoint molecules on the CTLs at day 6 and day 19 after adoptivetransfer. The expression patterns of each examined immune checkpointsare quite different. Both intrahepatic Akt1- and ctrl-engineered CTLs atday 6 after adoptive transfer express high level of PD-1 whenencountering the cognate antigen in the liver, but the PD-1 expressionis down regulated in the Akt1-CTLs at day 19 after adoptive transfer(FIGS. 4I-L).

At day 6 after exposure to HBV, a certain proportion of the hepaticAkt1-CTLs expressed high level of TIM-3, whereas splenic CTLs andctrl-CTLs in liver express lower level of TIM-3 at this time point,which suggests a stronger TCR triggering in Akt1-CTLs than in ctrl-CTLs(FIGS. 4M and 4N). However, during day 6 to day 19, the expression ofTIM-3 decreases in hepatic Akt1-CTLs, whereas it increases dramaticallyin the ctrl-CTLs in liver but not in the CTLs in spleen (FIGS. 4M-P).

Hepatic ctrl-CTLs express high level of LAG-3 at both day 6 and day 19after adoptive transfer, whereas Akt1-CTLs express less LAG-3 on theirsurface during the whole period (FIGS. 4R-T). Akt2-CTLs also showdramatic down-regulation of PD-1, TIM-3 and TIGIT (FIGS. 6A-6F).

These in-vitro and in-vive data clearly demonstrate that Akt signalingpossesses very few influence on PD-1 expression but positively regulatesTIM-3 expression on CTLs during early TCR signaling. We further provethat augmentation of Akt signaling prevents the expression of LAG-3 andTIGIT on CTLs in the liver during persistent HBV infection, which maycontribute the robust expansion and potent effector functions ofAkt-CTLs against HBV.

The higher expression of PD-1 and TIM-3 on Akt-CTLs than on ctrl-CTLsafter re-stimulation in vitro and in vivo strongly suggests a strongerTCR triggering in Akt-CTLs than that in ctrl-CTLs and also excludes thelack of antigen stimulation at this early time point, which results indown-regulation of LAG-3 and TIGIT. The early expression of TIM-3 onAkt-CTLs may additionally involve in the augmentation of effectorfunctions of Akt-CTLs to combat HBV infection. The reduced expression ofimmune checkpoints on Akt-engineered CTLs at the later time point mayresult from the lack of antigen stimulation due to the intense effectorfunctions of Akt-CTLs, which facilitates the early removal of the HBVantigen from the liver.

Example 5: Akt Signaling in CTLs Enhances their Effector Functions andFacilitated HBV Clearance

The cell number of adoptively transferred ctrl- or Akt1-engineeredHBc₉₃₋₁₀₀-specific CTLs in the liver and in the spleen of HBV carriermice is measured, and there are more Akt1-CTLs than ctrl-CTLs recoveredfrom the liver at both of day 6 and day 19 after adoptive transfer(FIGS. 7A-C).

Akt1-CTLs but not ctrl-CTLs eliminate persistent HBV infection within 14days after being adoptive transferred into HBV carrier mice (FIG. 7D).These Akt1-CTLs have better cytotoxic functions than ctrl-CTLs, which isrevealed by the elevated serum ALT level from day 3 to day 7 (FIG. 7E).The Akt1-CTLs are mainly in the liver rather than the spleen at day 6post adoptive transfer and dispersed to the spleen after antigenclearance (FIGS. 7B and 7C). From the H&E staining of the liversections, a huge number of mononuclear cells in the liver sinusoid ofmice receiving Akt1-CTLs at day 6 are observed after adoptive transfer(FIG. 7F).

Immunohistochemical staining is performed to visualize the HBcAg orcleaved caspase 3 expression by hepatocytes and immune cells in theliver of HBV carrier mice. There are less HBcAg-positive hepatocytes butmore cleaved caspase 3-positive apoptotic hepatocytes detected in theliver of mice receiving Akt1-CTLs than in the liver of mice receivingctrl-CTLs at day 6 after adoptive transfer (FIGS. 7G and 7H). Theapoptotic hepatocytes or HBcAg⁺ hepatocytes are surrounded bymononuclear cells in the liver of mice receiving Akt1-CTLs whichsuggests a cytotoxic role of these Akt1-CTLs against HBV-infectedhepatocytes (FIGS. 7G and 7H). There are more Gr-1⁺ myeloid cells andadoptively transferred CTLs (CD45.1⁺) detected in the liver of micereceiving Akt1-CTLs than in the liver of mice receiving ctrl-CTLs at day6 (FIGS. 7I and 7J).

After clearance of antigen, the liver histology appears back to normal,the mononuclear cells reduce and HBcAg-positive hepatocytes as well ascleaved caspase 3-positive hepatocytes are no longer detected in theliver of mice receiving Akt1-CTLs (FIGS. 7K-M). The number of Gr-1⁺myeloid cells also reduces whereas a significant number of CD45.1⁺adoptively transferred CTLs still exists in the liver of mice receivingAkt1-CTLs (FIGS. 7N and 7O). The ctrl-CTLs fail to clear HBV (FIGS. 7D,7G and 7L) and cannot induce significant inflammation after beingadoptively transferred into HBV carrier mice (FIGS. 7E-7O).

Akt2-CTLs also expand vigorously when encountering the cognate antigenin vive (FIGS. 8A and 8B), prevent T-cell exhaustion (FIGS. 6A-6F),exhibited strong cytotoxic function (FIG. 5C) and are more efficient toclear HBV infection than ctrl CTLs (FIG. 8D). Akt1- and Akt2-CTLs arefound more capable to produce IFN-γ and TNF-α than ctrl-CTLs after exvivo re-stimulation with the specific HBc peptide (FIGS. 9A-D), which isconsistent with their capability to induce inflammatory responses asseen in FIGS. 7A-7O.

Example 6: Akt1 Drives Only TCR Signaling-Dependent Expansion andFacilitates the Self-Renewal of CTLs

We further examined the capability of the engineered CTLs to eliminateantigen from the liver through the measurement of the bioluminescence inthe liver of the recipient mice. The loss of bioluminescence representedthe clearance of antigen from the liver. We found that Akt1-OT-I CTLswere more efficient than ctrl OT-I CTLs to eliminate OVA from the liver(FIG. 10A). They cleared the antigen within 7 days, which was also thepeak of the expansion of the cell population in the liver (FIGS. 10B and10C). These Akt1-OT-I CTLs were more capable to execute cytotoxicitytoward OVA-expressing hepatocytes than ctrl CTLs did, which was revealedby the elevated serum ALT level of mice receiving Akt1-CTLs at day 7post adoptive transfer (FIG. 10D).

Being concerned about that the overexpression of Akt molecules in CTLsmay potentially induced oncogenic property of the transduced cells, wetherefore monitored the numbers of intrahepatic and splenic transferredCTLs and serum ALT levels in the mice receiving ctrl-CTLs and Akt1-CTLsfor a longer period of time. The serum ALT levels of mice receivingAkt1-CTLs decreased to normal levels after the clearance of antigens andcell numbers of Akt1-CTL also dropped at least 5000-fold from day 7 today 63 (FIGS. 10D-F). We detected a lot of mononuclear cells lying inthe liver sinusoid of mice receiving Akt1-CTLs but not ctrl-CTLs at day7 post adoptive transfer (FIG. 10G). The architecture of the livers ofmice receiving Akt1-CTLs returned to normal at day 32 and day 63 afterclearance of antigen (FIG. 10G).

We further analyzed the proliferation capability of these adoptivelytransferred Akt1-CTLs or endogenous CD8⁺ T cells at day 7 and day 63,respectively and found that even in the absence of antigen, theAkt1-CTLs could still undergo higher grade DNA synthesis to sustainself-renewal than endogenous CD8⁺ T cells did, which explained themaintenance of the cell number after clearance of antigen (FIGS. 10H and10I). These Akt1-CTLs in the liver sinusoid were all Ki-67-positive atday 7 after adoptive transfer, which demonstrated that they wereundergoing vigorous proliferation and were barely detected in the liversinusoid at day 32 and day 63 after adoptive transfer (FIG. 10J).

Example 7: Akt Signaling Facilitates Development of T Cell Memory

It has been shown that virus-infected hepatocytes were highly sensitiveto CTL-induced cytotoxicity. The liver microenvironment after HDI maynot completely mimic that during viral infection. We thereforeestablished an adenovirus (Ad-Albp-OL)-based liver infection mouse modelwith persistent expression of OVA and luciferase only in the liver underthe transcriptional control of albumin promoter in order to study thefunctions of Akt in CTLs under the circumstance of intrahepaticpersistent viral infection. We first titrated the viral doses forinfection and found that infection with 2×10⁸ and 4×10⁸ iu ofAd-Albp-OL, respectively, could induce stable expression of luciferasefor more than 2 months (FIGS. 11A-11B). We then infected mice with 4×10⁸iu of Ad-Albp-OL, adoptively transferred Akt- and ctrl-CTLs,respectively, into the mice and performed several analyses following theexperimental scheme showed in FIG. 12A.

Similar to the data from HDI model, there were more Akt1- or Akt2-CTLsthan ctrl-CTLs detected in the liver and in the spleen ofAd-Albp-OL-infected mice at day 7 after adoptive transfer (FIG. 13A).The inflammation induced by Akt1- or Akt2-CTLs further promoted theinnate immune cell response. We could detect more CD11b⁺ myeloid cells,natural killer (NK) cells but not NK T cells in the liver of the micereceiving Akt-CTLs at day 7 after adoptive transfer (FIGS. 13B-D). Themice receiving Akt1-OT-I CTLs showed elevated ALT levels at day 7 andday 14 after the adoptive transfer of T cells and also cleared virusesat day 7 (FIGS. 12B and 12C). The mice receiving control OT-I CTLs didnot show ALT elevation nor viral clearance after the adoptive transfer(FIGS. 12B and 12C). At day 60 after adoptive transfer, the mice werere-challenged by HDI of pENTRY-OL or pENTRY vector as HDI control toexamine whether they developed antigen-specific T-cell memory. The micereceiving Akt1-CTLs showed mild liver damage as revealed by the ALTelevation during day 4 to day 7 after re-challenge. The ALT level inthese mice was much less than that in their primary response (FIG. 12B).The mice receiving Akt1-OT-I CTLs re-expressed antigen as revealed byluciferase activity at day 61 and rapidly eliminated antigen within 3days whereas the mice receiving ctrl-OT-I CTLs could not eliminateantigen after re-challenge (FIG. 12C).

Similar result was observed in the mice receiving Akt2-CTLs (FIGS. 13Eand 13F). We could detect antigen-specific T-cell expansion in the liverof mice receiving Akt1-CTLs at day 7 after re-challenge (FIG. 12D). Theliver histological examination showed that both the mice receiving ctrl-and Akt1-CTLs, respectively, had no obvious inflammation in the liver ofmice after re-challenge (FIG. 12E). However, we could detect more CD8⁺ Tcells as well as Gr-1⁺ myeloid cells in the liver sinusoid of the micereceiving Akt1-CTLs after re-challenge (FIGS. 12F and 12G). These datasuggest the Akt-engineered CTLs don't only harbor strong effectorfunctions but also are more efficient to develop T-cell memory and couldeliminate antigen rapidly when re-encounter the antigen. During primaryand re-call responses, we observed the recruitment of innate immunecells to the liver, which may be a reflection of tissue damage and forthe purpose of tissue repair. It is also possible that the Gr-1⁺ myeloidcells contribute to the expansion of CTL population during the primaryand re-call responses.

Example 8: Akt Signaling in CTLs Enhances their Cytotoxic Function andFacilitates Tumor Killing

The capability of Akt-engineered CTLs in killing of hepatocellularcarcinoma (HCC) is further examined and demonstrated that the tumorantigen-specific Akt2-engrafted CD8⁺ CTLs can accumulate in the tumorsites as well as in the liver at day 10 after adoptive transfer intoHCC-bearing mice (FIG. 14A). These Akt2-CTLs change the tumormicroenvironment and attract or activate the surrounding F4/80⁺macrophages in tumor sites (FIG. 14B).

A lot of cleaved caspase 3-positive tumor cells are detected in the micereceiving Akt2-CTLs but not in ctrl mice (FIG. 14C). Serum ALT iselevated in the mice receiving Akt2-CTLs starting from day 3 afteradoptive transfer but not in ctrl mice (118.1 U/L vs. 22.8 U/L). Thelevel of ALT in mice receiving Akt2-CTLs is continuously increasing atleast until day 10 after adoptive transfer (590.5 U/L).

Ctrl-, Akt1- and Akt2-engineered HBc₉₃₋₁₀₀-specific CTLs are adoptivelytransferred into HCC-bearing mice, respectively. The oncogenes-inducedHCC mouse model is engineered to express luciferase and surrogate tumorantigen-HBc₉₃₋₁₀₀ peptide in the tumor. The tumor growth can bemonitored using IVIS and demonstrate that Akt2- but not ctrl- orAkt1-CTLs effectively eliminate HCC as shown by the reduction of in vivobioluminescence and the disappearance of tumor nodules in the livers ofmice receiving Akt2-CTLs (FIGS. 15A-D).

It can be concluded that Akt2 activation enables CTLs to have strongeffector functions to kill tumor cells in the liver.

Example 9: Anti-Tumor Capability of Akt-Engineered Chimeric AntigenReceptor (CAR) T Cells

To further explore the potential application of Akt molecules on cancerimmunotherapy, plasmids carrying human or mouse Akt1 or Akt2 genes areconstructed and the ORF encoding anti-CEA chimeric antigen receptor(CAR) (FIG. 16A). The construction of the recombinant anti-CEA chimericantigen receptor used in this present invention were described inHombach et al. (Hombach, A.; Wieczarkowiecz, A.; Marquardt, T.; Heuser,C.; Usai, L.; Pohl, C.; Seliger, B.; Abken, H., Tumor-specific T cellactivation by recombinant immunoreceptors: CD3ζ signaling and CD28costimulation are simultaneously required for efficient IL-2 secretionand can be integrated into one combined CD28/CD3 signaling receptormolecule. J Immunol 2001, 167 (11), 6123-31). Activated mouse CD3⁺ Tcells are modified by recombinant retroviruses carrying mouse AKT1 gene,anti-CEA CAR ORF or both and then are monitored for their proliferationcapability, cytokine production and cytotoxicity.

The modified CTLs are co-cultured with a colorectal adenocarcinoma cellline with the expression of CEA, LS174T and the proliferation of theCTLs is monitored through detection of incorporation of a thymidineanalog, EdU. Both CD4⁺ and CD8⁺ T cells with the engraftment of anti-CEACAR can respond to stimulation of LS174T and proliferate. Akt signalingfurther enhances the proliferative capability of anti-CEA CAR-engraftedCD4⁺ and CD8⁺ T cells (FIG. 16B).

Higher levels of IL-2 and IFNγ are detected in the culture medium ofco-culture of LS174T cell line with T cells expressing anti-CEA CAR andAkt1 or Akt2 molecules compared that of T cells expressing solelyanti-CEA CAR (FIGS. 16C-F). Intracellular staining of IFNγ and granzymeB of the CD8⁺ T cells co-cultured with LS174T cells also proves thatAkt1 or Akt2 overexpression can enhance the cytokine production andcytotoxicity in CTLs (FIGS. 16G-J).

Akt1-overexpressing and Akt2-overexpressing CTLs are shown to have thecapability to overcome the proliferative arrest induced bymyeloid-derived suppressor cells (MDSCs) (FIGS. 16K and 16L), whichstrongly suggests that the potential application of Akt molecules onT-cell engineering technology e.g. CAR T cells for immunotherapy.

This present invention provides a method able to enhance survival andfunctionality of anti-tumor or anti-viral T cells through overexpressionof Akt molecules in CTLs. The Akt-overexpressing CTLs are shown to havehigh proliferation capability and superior effector functions duringencounter with the antigen in the liver, which suggests that the Aktmolecules can help the CTLs to overcome T-cell exhaustion in theinhibitory microenvironment. This present invention further showsexpression of Akt molecules can facilitate anti-viral and anti-tumor CTLresponses e.g. proliferation, cytokine production and cytotoxicity.Moreover, it enables the CTLs resistance to proliferative arrest inducedby MDSCs. To sum up, the expression of constitutively active Aktmolecules enable T cells to gain the privilege to survive and to kill inthe tolerogenic liver or tumor microenvironments. The active Aktmolecules only when in combination with TCR signaling can triggermassive proliferative response of CTLs and therefore are safe to beapplied to T-cell engineering of CTLs. Inventors therefore have thefollowing claims for the compositions comprising the anti-tumor oranti-viral engineered T cells and the methods using thereof fortreatment of chronic viral infections and malignancies.

1. A composition for reducing immune tolerance which comprising anengineered cell overexpressing Akt molecules, the engineered cell isengineered with a polynucleotide encoding: a. an Akt isoform; and b. apeptide leading the Akt isoform to cell membrane of the engineered cell.2. The composition according to claim 1, wherein the Akt isoform isselected from the group consisting of Akt1, Akt2, and Akt3, or acombination thereof.
 3. The composition according to claim 1, whereinthe peptide is a myristoylation-targeting sequence set forth in SEQ IDNO:
 7. 4. The composition according to claim 1, wherein thepolynucleotide further comprising a fragment encoding a chimeric antigenreceptor or a recombinant T cell receptor.
 5. The composition accordingto claim 4, wherein the polynucleotide further comprising a fragmentencoding a linker between the Akt isoform and the chimeric antigenreceptor or the recombinant T cell receptor.
 6. The compositionaccording to claim 5, wherein the linker is a 2A peptide set forth inSEQ ID NO:
 9. 7. The composition according to claim 1, wherein theengineered cell is a T cell, a nature killer cell, a hematopoietic stemcell, an embryonic stem cell or a pluripotent stem cell.
 8. A method fortreating a virus infection disease in a subject comprising administeringto the subject an effective amount of the composition according toclaim
 1. 9. The method according to claim 8, wherein the virus infectiondisease is hepatitis.
 10. A method for treating a cancer in a subjectcomprising administering to the subject an effective amount of thecomposition according to claim
 1. 11. The method according to claim 10,wherein the cancer is a liver cancer.
 12. The method according to claim11, wherein the liver cancer comprising hepatocellular carcinoma, bileduct carcinoma, hepatic angiosarcoma and epithelioidhemangioendothelioma.
 13. A method for treating a cancer in a subjectcomprising administering to the subject an effective amount of thecomposition according to claim
 4. 14. A method for producing thecomposition according to claim 1, which comprising transferring arecombinant viral or transposon vector into a target cell, and expandingthe target cell.
 15. The method according to claim 14, wherein therecombinant viral or transposon vector can be a retrovirus, alentivirus, an adenovirus, an adeno-associated virus, or other relatedviruses and various transposon systems can be used in transduction orintegration of transgenes.
 16. The method according to claim 14, whereinthe recombinant viral or transposon vector can be amplified throughplasmid amplification, in vitro transcription or in vitro synthesis andtransfected into the target cell through electroporation, liposome orother chemical vehicles.
 17. The method according to claim 14, whereinthe target cell can be a T cell, a nature killer cell, a hematopoieticstem cell, an embryonic stem cell or a pluripotent stem cell.
 18. Themethod according to claim 14, wherein the target cell can be furthermodified by viral transduction and DNA or RNA transfection.
 19. Themethod according to claim 14, wherein expanding the target cellcomprising stimulating the target cell with soluble, plate-boundanti-CD3 and anti-CD28 antibodies or with anti-CD3 and anti-CD28 beadswith supplement of cytokines to enhance the growth of the target cell.